If glucagon plays a hormonal role in the regulation of gluconeogenesis from endogenous amino acids, its secretion might be stimulated by an increase in the concentration of alanine, which has recently been identified as a principal gluconeogenic precursor. To determine if this is the case, 0.75 mmole of alanine per kilo was infused into conscious dogs immediately after a priming injection of 0.25 mmole per kg for 15 min. A uniform rise in the plasma level of pancreatic glucagon, as determined by a relatively specific radioimmunoassay for pancreatic glucagon, was observed. The rise, which averaged 90 pg per ml, was highly significant at 7(1/2) and 15 min after the start of the infusion. Insulin rose an average of only 8 muU per ml, while glucose rose an average of 10 mg per 100 ml. A lower dose of alanine, 1 mmole per kg, infused over a 1 hr period without an initial priming injection, also elicited a significant rise in glucagon measured in the pancreaticoduodenal venous plasma; glucagon rose from 350 pg per ml to 1066 pg per ml at the end of the infusion. The insulin response was modest and inconsistent, and glucose, again, rose 10 mg per 100 ml. To determine if the availability of exogenous glucose would abolish the alanine-induced rise in glucagon secretion, dogs were made hyperglycemic by a constant intravenous glucose infusion and were then given the high-dose alanine infusion. Under these circumstances, glucagon did not rise above the mean fasting concentration of 75 pg per ml, whereas mean insulin rose dramatically by more than 100 muU per ml. It was concluded that, in the fasting state, alanine does stimulate the secretion of glucagon, while having very little stimulatory effect on insulin secretion. Glucagon could, therefore, be a humoral mediator of gluconeogenesis from endogenous alanine, responding to hyperalaninemia in the fasting state, but not when exogenous glucose is available.